Amity Institute of Biotechnology, Amity University Haryana, Gurugram, Manesar, 122413, India.
Amity Lipidomics Research Facility, Amity University Haryana, Gurugram, Manesar, 122413, India.
Microb Pathog. 2020 Dec;149:104262. doi: 10.1016/j.micpath.2020.104262. Epub 2020 May 18.
Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) has emerged in recent decades as one of the leading causes of mortality worldwide. The burden of TB is alarmingly high, with one third affected global population as reported by WHO. Short-course treatment with an antibiotic is a powerful weapon to treat infection of susceptible MTB strain, however; MTB has developed resistance to anti-TB drugs, which is an escalating global health crisis. Thus there is urgent need to identify new drug targets. RecA is a 38 kilodalton protein required for the repair and maintenance of DNA and regulation of the SOS response. The objective of this study is to understand the effect of disruption of RecA gene (deletion mutant ΔdisA from previous study) in a surrogate model for MTB, Mycobacterium smegmatis. This study demonstrated that disruption of RecA causes enhanced susceptibility towards rifampicin and generation of ROS leading to lipid peroxidation and impaired membrane homeostasis as depicted by altered cell membrane permeability and efflux pump activity. Mass spectrometry based lipidomic analysis revealed decreased mycolic acid moieties, phosphatidylinositol mannosides (PIM), Phthiocerol dimycocerosate (DIM). Furthermore, biofilm formation was considerably reduced. Additionally, we have validated all the disrupted phenotypes by RT-PCR which showed a good correlation with the biochemical assays. Lastly, RecA mutant displayed reduced infectivity in Caenorhabditis elegans illustrating its vulnerability as antimycobacterial target. Together, present study establishes a link between DNA repair, drug efflux and biofilm formation and validates RecA as an effective drug target. Intricate studies are needed to further understand and exploit this therapeutic opportunity.
结核病（TB）是由结核分枝杆菌（MTB）引起的，近几十年来已成为全球主要死亡原因之一。世界卫生组织（WHO）报告称，全球有三分之一的人口受到结核病的影响，其负担令人震惊。用抗生素进行短程治疗是治疗易感 MTB 菌株感染的有力武器；然而，MTB 已经对抗结核药物产生了耐药性，这是一个不断升级的全球健康危机。因此，迫切需要确定新的药物靶点。RecA 是一种 38 千道尔顿的蛋白质，是修复和维持 DNA 以及调节 SOS 反应所必需的。本研究的目的是了解在结核分枝杆菌替代模型分枝杆菌中，RecA 基因（来自先前研究的缺失突变体ΔdisA）的破坏对其的影响。本研究表明，RecA 的破坏导致对利福平的敏感性增加，并产生 ROS，导致脂质过氧化和膜稳态受损，表现为细胞膜通透性改变和外排泵活性增强。基于质谱的脂质组学分析显示，分枝菌酸部分、磷酸甘露糖脂（PIM）和 Phthiocerol dimycocerosate（DIM）减少。此外，生物膜形成明显减少。此外，我们通过 RT-PCR 验证了所有破坏的表型，这与生化测定有很好的相关性。最后，RecA 突变体在秀丽隐杆线虫中的感染性显著降低，表明其作为抗分枝杆菌靶标很脆弱。综上所述，本研究建立了 DNA 修复、药物外排和生物膜形成之间的联系，并验证了 RecA 作为有效的药物靶点。需要进一步深入了解和利用这一治疗机会的复杂研究。
